Peptide antibiotics

ABSTRACT

Peptides of the formula ##STR1## wherein R is CH 3  --(CH 2 ) 6 , CH 3  --(CH 2 ) 4  --CH═CH--(CH 2 ) 2  and CH 3  (CH 2 ) 8  having antibiotic and antitumor activity are prepared by cultivation of the novel microorganism Polyangium brachysporum. Enzymatic hydrolysis of those peptides gives other peptides useful as intermediates in the preparation of peptides having activity as antibiotics and/or antitumor agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of our earlier co-pending applicationSer. No. 855,649, filed Apr. 25, 1986, now U.S. Pat. No. 4,692,510,which is a continuation-in-part of application Ser. No. 771,090, filedAug. 30, 1985, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel peptide antibiotics and to their use asantimicrobial and antitumor agents. The present invention also relatesto methods for the preparation of these antibiotics, intermediatestherefor and to the novel microorganism used in their preparation byfermentation.

Various microorganisms have been isolated from soil samples, cultured ina synthetic medium and found to elaborate products having antibioticactivity. We have isolated a novel bacterium species, K481-B101, from asoil sample collected near the Parthenon in Athens, Greece, anddiscovered it to produce a mixture of peptides having antibioticactivity.

It is a primary object of this invention to provide novel peptideantibiotics particularly useful as antifungal and antitumor agents.

It is also an object of the present invention to provide convenientmethods for the preparation of these antibiotics.

And, it is a further object of the invention to provide pharmaceuticalcompositions containing those antibiotics.

SUMMARY OF THE INVENTION

In accordance with the aforementioned objects, the present invention isa compound of the formula ##STR2## wherein R is CH₃ --(CH₂)₆, CH₃--(CH₂)₄ --CH═CH(CH₂)₂ or CH₃ --(CH₂)₈.

In a further aspect, the invention includes pharmaceutical compositionscomprising an amount of those compounds, singly or in combination,effective as an antifungal and/or antitumor agent, together with apharmaceutically acceptable carrier.

In its preparative aspect, the present invention is a method for thepreparation of a compound of the formula ##STR3## wherein R is CH₃--(CH₂)₆, CH₃ --(CH₂)₄ --CH═CH--(CH₂)₂ or CH₃ --(CH₂)₈, which comprisesculturing Polyangium brachysporum sp. nov. in a nutrient mediumcontaining an assimilable source of carbon and nitrogen under aerobicconditions, separating the mycelia produced and recovering the compoundfrom the nutrient medium.

In a further composition aspect, the present invention includes thecompounds ##STR4## useful as intermediates in the preparation of thecompounds of the invention, and methods for the preparation of thoseintermediate compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are the IR spectra of Bu-2867T A, B and C,respectively.

FIGS. 4 and 5 are the 'H-NMR and ¹³ C-NMR spectra of Bu-2867T A.

DETAILED DESCRIPTION

The morphological, cultural and physiological characterization ofK481-B101 was made by the methods described by McCurdy, Jr. H. D.:Studies on the Taxonomy of the Myxobacterales. II, Polyangium and thedemise of the Sorangiaceae. Intl. J. Syst. Bacteriol. 20: 283-296, 1970;Reichenbach, H.: Nannocystis exedens gen. nov., sp. nov., a newmyxobacterium of the family Sorangiaceae. Arch. Mikrobiol. 70: 119-138,1970; Christensen, P. and F. D. Cook: Lysobacter, a new Genus ofnon-fruiting, gliding bacteria with a high base ratio. Intl. J. Syst.Bacteriol. 28: 367-393, 1978; and Christensen, P.: Synonymy ofFlavobacterium pectinovorum Dorey with Cytophaga johnsona Stanier. Intl.J. Syst. Bacteriol. 27: 122-132, 1977. Maintenance and purification wasby the procedures described by Peterson, J. E.: Isolation, cultivationand maintenance of the myxobacteria. Methods in Microbiology 3B:185-210, 1969. Edit. J. R. Norris & D. W. Ribbons. Academic Press(London and New York); and Reichenbach, H & M. Dworkin: The OrderMixobacterales. The Prokaryotes. Volume I: 328-355, 1981. Edit. M. P.Starr et al. Springer-Verlag (Berlin, Heidelberg and New York). Thetaxonomic position was determined according to the descriptions inBergey's Manual, 8th Ed., 1974 and "The Prokaryotes, Vol. I".

Morphology:

Casitone-Mg⁺⁺ agar, chitin agar, yeast cell agar and rabbit dungpellet-water agar were used for the morphological study. K-481-B101 is aGram-negative, non-flagellate bacterium. The vegetative cells arecylindrical (0.6-0.8 by 2-10 micrometers) with blunt rounded ends. Thevegetative cells show flexible and slow gliding movements on moistsurface of agar medium or soft agar medium. Myxospores differ clearlyfrom vegetative cells, are oval or spherical, 0.6-0.8 by 0.6-1.5micrometers, non-refractile or refractile, and occasionally pair.K481-B101 forms on most descriptive agar media sessile sporangiaenveloping myxospores. The sporangia are oval, spherical or pillow-like,fairly variable in size, 12×20 to 80×120 micrometers, often bounded by acommon envelope or slimy layer, double contoured, and occur singly or inclusters (sori). The morphology of K481-B101 is represented in Table 1.

Cultural characteristics:

K481-B101 grows moderately on casitone-Mg⁺⁺ agar (McCurdy, 1969) andyeast cell agar (Christensen & Cook, 1978), but poorly on Bacto-nutrientagar or Bacto-heart infusion agar. Rhizoid or feathery swarmings areobserved on YP-soft agar (yeast extract 0.3%, peptone 0.1%, NaCl 0.01%,Agar 0.3%, pH 6.6-6.8), but not on casitone-Mg⁺⁺ agar. The colonies oncasitone-Mg⁺⁺ agar are circular, translucent and pale greenish yellow,and weakly etch, erode or penetrate into agar. The culturalcharacteristics are shown in Table 2.

Physiological characteristics:

K481-B101 hydrolyzes starch, chitin, gelatin and casein, but notcellulose and agar. It lyses autoclaved yeast cell, but not living cellof Micrococcus luteus. K481-B101 is mesophilic, and sensitive to 2%NaCl. The physiological characteristics are shown in Table 3.

Concomitance of flagellate bacteria and occurrence of spontaneousvariant:

Concomitance of Gram-negative, rod-shaped flagellate bacteria wasobserved in the original culture. K481-B101 was fairly well purified bycombining the usual techniques of dilution and single cell isolationwith sonication, heat shock treatment or antibiotic sensitivity (e.g.pipemidic acid at 50 mcg/ml) using the myxospore or fruiting body.Unpurified culture of K481-B101 occurred mucoid variants which formwhitish dome-shaped colony with swarming halo. The vegetative cells ofthese mucoid variants are somewhat larger than the parental strain, andmeasured 0.8-1.0×2.0-3.5 micrometers. The cluster of sporangia (sorus)is predominantly formed by mucoid variants.

Taxonomic position:

K481-B101 is a fruiting gliding bacterium, isolated from a soil sample.The diagnostic major characteristics of the strain are as follows:

Vegetative cells:

(1) cylindrical, of uniform diameter

(2) not tapered at ends

(3) penetrable into agar media

(4) Congo red, not adsorbed

Myxospores:

(1) differentiated from vegetative cells

(2) oval or spherical

(3) non-refractile or refractile

Sporangia:

(1) sessile

(2) oval, spherical or irregular

(3) often bounded by a common envelope or slimy layer

(4) double contoured

(5) pale yellow (lack of distinct color)

(6) occurring singly or in clusters (sori)

Cultural and physiological characteristics:

(1) colony, golden yellow to whitish

(2) colonies, weakly etch, erode and penetrate agar

(3) chitinolytic but not cellulolytic

(4) yeast cell lyzed, but Micrococcus luteus not lyzed

The above-mentioned morphological, cultural and physiologicalcharacteristics of K481-B101 indicate that K481-B101 is classified intothe order Myxobacterales. Among the genera of Myxobacterales, the generaMyxococcus, Archangium and Cystobacter are differentiated from K481-B101on account of the tapered vegetative cells and the fruiting bodymorphology. The genera Melittangium, Stigmatella and Chondromyces differfrom K481-B101 in the stalked sporangia.

K481-B101 is similar to the genera Polyangium and Nannocystis. K481-B101resembles the genus Nannocystis in the formations of oval or sphericalmyxospores, and oval or spherical, solitary sporangia, but differs fromthe latter in the cylindrical vegetative cells of uniform diameter andthe lack of ability to etch, erode and penetrate into agar. K401-B101resembles the genus Polyangium in the cylindrical vegetative cells withblunt rounded ends, the predominant formation of non-refractilemyxospores and the oval or spherical, double contoured sporangia. Basedon the results of comparative studies with the genera of OrderMyxobacterales, K481-B101 is considered to be classified as a species ofthe genus Polyangium. Among the species of Polyangium, P. luteum issimilar to K481-B101 in the size of vegetative cells, the color andshape of sporangia and the color of vegetative colony. However,K481-B101 differs from P. luteum in the oval or spherical myxosporeswhich are much contracted and the lack of ability to lyze bacterialliving cells such as the cells of Micrococcus luteus.

Thus, K481-B101 is concluded to be a new species of the genus Polyangiumin the family Polyangiaceae, the Order Myxobacterales, and is proposedto be designated Polyangium brachysporum sp., nov. The type strain isNo. K481-B101 (single isolate), and the culture which has been depositedin the American Type Culture Collection with the accession number ATCC53080.

                  TABLE 1                                                         ______________________________________                                        Morphology of K481-B101                                                       ______________________________________                                        Vegetative cells:                                                                         Gram-negative. Cylindrical with                                               blunt rounded ends, (0.6-0.8 by 2.0-                                          10 micrometers). Congo red, not ad-                                           sorbed                                                            Myxospores: Distinguishable from vegetative cells.                                        Much shrunken, becoming oval or                                               spherical, 0.6-0.8 by 0.6-1.5 micro-                                          meters, non-refractile. Longer                                                incubation affords refractile ones.                               Sporangia:  Sessile, occurring singly or in                                               clusters. Oval, spherical, pillow-                                            shaped or shapeless. Considerably                                             variable in size, 12 × 20 to 80 × 120                             micrometers. Bounded by a common                                              envelope or slimy layer. Double                                               contoured. Embedded in agar.                                                  Clusters of two to ten or more                                                sporangia range 50 to 300 micrometers                                         in size of total mass.                                            Microcolony:                                                                              On chitin agar after incubation for 2                                         weeks. Palisade or zigzag arrangement                                         of chains of vegetative cells at                                              periphery. Gliding movement of single                                         cells is observed, but that of cell                                           masses is not seen.                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Cultural characteristics of K481-B101 Colony                                  on casitone-Mg++ agar (McCurdy, 1969) at 28° C. for 6                  ______________________________________                                        days                                                                          Form:          circular                                                       Surface:       smooth, later partially wrinkled                               Elevation:     raised                                                         Edge:          entire or somewhat irregular, and                                             absence of distinct protrusion such                                           as shapes of tongue, feather or                                               rhizoid                                                        Optical property:                                                                            semi-transparent or opaque                                     Color of colony:                                                                             pale greenish yellow                                           Diffusible pigment:                                                                          none                                                           ______________________________________                                    

Growth on chitin agar after incubation at 28° C. for 3 weeks.

Thin, translucent, pale yellow or colorless. Thick, opaque andyellowish-white at peripheral part. Concentric formation of sporangia atthe periphery. Weakly etch, erode or penetrate the agar.

                  TABLE 3                                                         ______________________________________                                        Physiological characteristics of K481-B101                                    ______________________________________                                        Hydrolysis of                                                                 Soluble starch    +                                                           Potato starch     +                                                           CMC sodium        +                                                           Cellulose         -                                                           Agar              -                                                           Chitin            +                                                           Alginate sodium   -                                                           Polypectate sodium                                                                              -                                                           Gelatin           +                                                           Casein            +                                                           Growth on                                                                     Simon's citrate agar                                                                            -                                                           Christensen citrate agar                                                                        -                                                           Glucose-ammonium salts agar                                                                     -                                                           Asparagin-ammonium salts agar                                                                   +                                                           Production of                                                                 Indole            -                                                           H.sub.2 S         +                                                           Acetoin (VP-reaction)                                                                           -                                                           Urease            +                                                           Oxidase           +                                                           Catalase          +                                                           Lytic Action to                                                               Living cell of Micrococcus                                                                      -                                                           luteus strains PCI 1001 &                                                     ATCC 9341                                                                     Autoclaved yeast cell                                                                           +                                                           Reactions                                                                     Milk coagulation: -                                                           Milk peptonization:                                                                             +                                                           NaCl tolerance:   Growth: 1.0% NaCl or less                                                     No growth: 2.0% NaCl or                                                       more                                                        pH tolerance:     Growth range: pH 5.5-10.5                                                     Scant growth: pH 5.0                                                          No growth: pH 4.5 and                                                         11.5                                                        Growth temperature                                                                              Maximal growth: 37° C.                                                 Growth range: 15° C.-42° C.                                     No growth: 10° C. and 45° C.                  Oxidative or fermentative:                                                                      Oxidative                                                   reaction          (Hugh and Leifson medium)                                   ______________________________________                                    

Antibiotic Production:

The stock culture of Polyangium brachysporum K481-B101 was propagated at28° C. for 3 days on agar slant medium composed of 0.5% soluble starch,0.5% glucose, 0.1% meat extract, 0.1% yeast extract, 0.2% Nz-case, 0.2%NaCl, 0.1% CaCO₃ and 1.6% agar (pH 7.0). A well grown agar slant wasused to inoculate the vegetative medium consisting of 2% corn starch, 3%soybean meal, 0.3% MgSO₄. 7H₂ O and 1% CaCO₃ (pH 7.0, beforesterilizaton). After incubation at 28° C. for 3 days on a rotary shaker(250 rpm), 5 ml of the growth was transferred into a 500-ml Erlenmeyerflask containing 100 ml of the production medium having the samecomposition as the vegetative medium.

The antibiotic production was monitored by the paper disc agar diffusionmethod using Candida albicans A9540 as the test organism. Thefermentation was continued for 4 days at 28° C. on a rotary shaker andthe antibiotic production reached a maximum of 100 mcg/ml.

The fermentation was also carried out in a stir-jar fermenter. A 500-mlportion of the seed culture obtained by flask fermentation was used toinoculate 10 liters of the production medium in a 20-liter vessel. Thefermentation was carried out at 28° C. with agitation at 250 rpm andaeration at 10 liters per minute. The antibiotic production reached amaximum of 150 mcg/ml after forty hours' fermentation.

Isolation and purification of antibiotic:

The fermentation broth (48 L) was centrifuged with the aid of asharpless centrifuge. The mycelial cake was homogenized with 7 L ofmethanol and the mixture stirred for one hour. After removal of theinsolubles by filtration, the methanol extract was evaporated to anaqueous solution which was combined with the broth filtrate andextracted with n-butanol (24 L). The extract was concentrated to 0.5 Lwhich was poured into n-hexane (3.5 L) under stirring to precipitate thecrude antibiotic (41 g). This solid was chromatographed on a column ofsilica gel C-200 (760 ml) eluting with ethyl acetate and an increasingamount of methanol (0-50%). The bioactivity eluted was detected by apaper disc assay using Candida albicans A9540 as the test organism. Theactive fractions were combined and evaporated to yield pale yellowpowder (13 g) of BU-2867T complex. A 200 mg-portion of this solid waschromatographed on a reverse-phase column (C₁₈, 100 ml) usingethanol-water (3:7 to 5:5) as an eluant. The eluate was monitored byantifungal bioassay and TLC (Silanized, EtOH:H₂ O=55:45). The firstactive fractions were combined and evaporated under reduced pressure toafford pure white solid of BU-2867T A (61 mg) which was crystallizedfrom aqueous methanol to deposit colorless needles (34 mg). Evaporationof the second and third active fractions yielded BU-2867T B (1 mg) and C(11 mg), respectively. BU-2867T C was crystallized from methanol as fineneedles. Repetition of the above reverse-phase chromatography afforded atotal of 3.9 g of BU-2867T A, 44 mg of BU-2867T B and 342 mg of BU-2867TC.

Characterization of antibiotic:

BU-2867T A and C were isolated as colorless needles while BU-2867T B wasobtained as white amorphous powder. They are readily soluble inmethanol, ethanol, n-butanol and dimethyl sulfoxide, slightly soluble inchoroform, acetonitrile and ethyl acetate and practically insoluble inn-hexane and water. They gave a positive response to Rydon-Smith reagentand colored upon spraying of iodine or sulfuric acid on TLC plate. Theywere negative to ninhydrin, Sakaguchi, anthrone and Dragendorffreaction. BU-2867T A, B and C were analyzed for C₂₇ H₄₄ N₄ O₆, C₂₉ H₄₆N₄ O₆ and C₂₉ H₄₈ N₄ O₆, respectively, by the mass spectrometry andmicroanalyses. The UV spectra of the three components in methanolexhibited a single maximum at 261 nm, which did not shift in acidic oralkaline solution. The physico-chemical data of BU-2867T A, B and C aresummarized in Table 4. Their IR spectra in KBr (FIGS. 1, 2 and 3) showedstrong amide bands at around 1630 and 1540 cm⁻¹ and OH and/or NHabsorption at 3300 cm⁻¹. The ¹ H-NMR spectrum of BU-2867T A (FIG. 4)revealed the presence of six olefinic protons (δ:6.16, 6.20 (2H), 6.28,6.49 and 7.09 ppm) and four amide protons (δ:7.49, 7.82, 7.87 and 8.72ppm). Two methyl groups (δ:0.93 ppm, t and 1.07 ppm, d) were alsoobserved in the spectrum. The ¹³ C-NMR spectrum of BU-2867T A (FIG. 5)showed more than 23 carbon signals including four carbonyl carbons, sixolefinic carbons and three methyl carbons. BU-2867T B and C showed the¹³ C-NMR spectra very similar to that of BU-2867T A except the presenceof two more olefinic carbons in BU-2867T B and two more methylenecarbons in BU-2867T C than in BU-2867T A.

BU-2867T A was heated under reflux with 6N HCl for 16 hours. Afterremoval of the lipophilic product (V) by ethyl acetate extraction, thehydrolyzate was concentrated to an oily residue which is chromatographedon Dowex 50W×4 ion-exchange resin by developing with pyridine-formicacid-acetic acid buffer (0.1-0.2M, pH 3.1-5.1). By monitoring withninhydrin reagent, four amino acids I, II, III and IV eluted in thatorder were isolated and crystallized as hydrochlorides. Amino acid I([α]_(D) 25° :-12.7° in 5N HCl) was identified as L-threonine by itsphysicochemical properties. The ¹ H-NMR spectrum and EI-MS (M⁺ +1:m/z134) of amino acid II indicated that it was a mixture ofdiastereoisomers of 4-amino-3-hydroxy-n-valeric acid, Konishi, M.; K.Saito, K. Numata, T. Tsuno, K. Asama, H. Tsukiura, T. Naito & H.Kawaguchi: Tallysomycin, a new antibiotic complex related to bleomycin.II. Structure determination of tallysomycins A and B. J. Antibiotics 30:789-805, 1977, and its identity was confirmed by a direct comparisonwith the authentic sample. The molecular formula of III was assigned tobe C₅ H₉ NO₂ by elemental analysis and EI-mass spectrometry (M⁺ : m/z115). Its ¹ H-NMR showed the presence of one methyl (δ:1.50 ppm, d,J:6.0 Hz), one methine (δ:4.0-4.2, m) and two trans olefinic protons(δ:6.05 ppm, d, J:15.0 Hz and 6.57 ppm, d-d, J:6.0 and 15.0 Hz). Thesespectral data clearly indicated III to be 4-amino-2(E)-pentenoic acid,Honore', T.; H. Hjeds, P. Krogsgaard-Larsen & T. R. Christiansen:Synthesis and Structure-Activity Studies of Analogs of γ-amino-butyricAcid (GABA). Eur. J. Med. Chem., 13:429-434, 1978. 2(S)-Configurationwas assigned to III based on its specific rotation ([α]D²².5 :-6° in 5NHCl). Amino acid IV was determined to be 4-hydroxylysine based on itselemental analysis (C₆ H₁₄ N₂ O₃) EI-MS (M +1 : m/z 163) and ¹ H-NMRspectrum (δ:1.8-2.1 ppm 4H, m, 3.18 ppm, 2H, t and 3.6-4.3 ppm, 2H, m)and the formation of a γ-lactone compound (.sup.υ C═O : 1770 cm⁻¹) upontreatment with 6N HCl. Izumiya, N.; Y. Fujita, F. Irreverre & B. Witkop:The Synthesis of Erythro-γ-hydroxy-L-lysine and its Nonoccurrence inCollagen. Biochemistry 4:2501-2506, 1965, reported mutarotation of4-hydroxy-lysines and the shift observed for IV indicatederythro-L-configuration. Thus, the structure of amino acid IV wasestablished to be erythro-4-hydroxy-L-lysine.

The lipophilic acidic fraction (V) obtained in the above acid hydrolysiswas treated with diazomethane to yield an oily methyl ester (VI) afterchromatographic purification. Its UV (λ_(max) MeOH: 260 nm, ε: 22,000),EI-MS (M⁺ : m/z 210) and ¹ H-NMR (four --CH═, one C--CH₃ and six--CH₂₋₋) indicated that VI was methyl 2,4-dodecadienoate. The magnitudeof coupling constant made it obvious that both double bonds had a transgeometry. The original acid V was thus 2(E), 4(E)-dodecadienoic acid,Burden, R. S. & L. Crombie: Amides of Vegetable Origin, Part XII. A newseries of alka-2,4-dienoic tyramine-amides from Anacyclus pyrethrum D.C.(Compositae). J. Chem. Soc. (C), 1969: 2477-2481, 1969. ##STR5##BU-2867T A has the molecular formula of C₂₇ H₄₄ N₄ O₆ and showed sixolefinic carbons and four amide carbons in the ¹³ C-NMR. It wastherefore apparent that amino acid II was an artifact produced byhydration of the natural amino acid III during acid hydrolysis. Theantibiotic should be a cyclic peptide since it was negative to ninhydrinreaction and did not exhibit any titratable function in potentiometrictitration. This was further supported by a fact that BU-2867T A affordeda di-O-acetyl derivative (EI-MS : M⁺ =m/z 604, ¹ H-NMR: 2.02 ppm, 3H and2.08 ppm, 3H) upon acetylation in pyridine. The MS of the acetate alsoindicated strong fragment ions at m/z 179 and 322 which suggested thepresence of V→I→ sequence in the antibiotic.

BU-2867T A was subjected to enzymatic degradation with ficin in 0.01Mphosphate buffer (pH 7.0). After being acidified to pH 2.2, the reactionmixture was extracted with n-butanol to obtain an acidic compound (VII).Subsequent extraction of the aqueous solution at pH 10.0 with n-butanolafforded a ninhydrinpositive substance (VIII). Compound VII was shown tobe 2,4-dodecadienoyl-threonine (V→I) by the IR (.sup.ν_(C)═O :1720 &1650 cm⁻¹), EI-MS (M⁺ -H₂ O : m/z 279 and M⁺ -THR : m/z 179) and ¹ H-NMRspectrum. The structure was further substantiated by the fact that VIIyielded I and V on hydrolysis in 6N HCl. When refluxed in 6N HCl,compound VIII afforded amino acids II, III and IV as revealed by TLC. Inthe EI-MS of VIII, the molecular ion was found at m/z 241 indicating acyclic peptide structure. The ¹ H-NMR (270 MHz, in DMSO-d₆) exhibitedtwo amide NH protons at: 7.43 ppm (triplet) and 9.10 ppm (broad) inagreement with the above cyclic structure. Extensive decouplingexperiment revealed that the amino group of III and ε-amino group of IVwere linked to carbonyls to form amides while α-amino group of IV wasfree. Thus, the structure of VII and VIII were assigned as shown below:##STR6##

Compounds VII and VIII were also obtained when BU-2867T A was subjectedto enzymatic degradation or hydrolysis with papain or chymopapain. Amixture of BU-2867T A (4 g) and papain (Sigma P-3375, 50 g) in 20 L of10% aqueous methanol was stirred at 28° C. for 22 hours. The mixture wasthen acidified to pH 3.3 by acetic acid and extracted with ethyl acetate(10 L). Evaporation of the extract afforded an oil (6.3 g) which waschromatographed on silica gel (Wakogel C-200, 250 ml) with a mixture ofCH₂ Cl₂ and methanol (9:1) to give a semi-pure, oily compound VII (3.3g). Further chromatography of the oil by Sephadex LH-20 and subsequentcrystallization gave colorless needles of pure VII. 1.15 g (yield 50%).M.p. 90°-91° C. [α]_(D) ²⁷ +17.4° (C 0.5, MeOH), Anal. Calcd. for C₁₆H₂₇ NO₄.H₂ O : C 60.93, H 9.27, N 4.44. Found: C 61.34, H 9.03, N 4.20.EI-MS: m/z 279 (M⁺ -H₂ O). UV λmax^(MeOH) nm (ε): 260 (28,000). IR.sup.υ max (KBr) cm⁻¹ : 3520, 3410, 3300, 1720, 1690, 1655, 1630, etc. ¹H-NMR (80 MH_(z), DMSO-d₆) δ 0.88 (3H, t), 1.06 (3H, d), 6.20 (3H, m),7.00 (1H, m), 7.84 (1H, d, NH), etc.

After ethyl acetate extraction, the acidic aqueous solution wasconcentrated to dryness. The residue (36 g) was dissolved in 50 ml ofwater, adjusted to pH 9.0 and applied on a column of reverse phasesilica (C₁₈, Merck, 1.6 L) which was developed with water. The fractionscontaining compound VIII were pooled and concentrated in vacuo. Theresidue was chromatographed on Sephadex LH-20 (250 ml) with 50% aqueousmethanol and then on reverse phase silica (C₁₈) with acidic water (pH3.0 by dil HCl) afforded pure VIII hydrochloride. 747 mg (yield 35%).M.p. 190° C. (dec.). [α]_(D) ²⁶ -113° (c 0.5, H₂ O). EI-MS : m/z 241(M⁺). UV: end absorption. IR υ_(max) (KBr) cm⁻¹ : 3400, 1660, 1620,1530, etc. ¹ H-NMR (80 MH_(z), DMSO-d₆) δ 1.27 (3H, d), 1.4-1.8 (4H),2.98 (2H, m), 4.52 (1H, m), 6.19 (1H, d), 6.45 (1H, d-d), 7.43 (1H, t,NH), 9.46 (1H, d, NH). Analysis Calcd for C₁₁ H₉ N₃ O₃.HCl.H₂ O : C44.67, H 7.50, N 14.21, Cl 11.99. Found: C 45.04, H 7.82, N 13.81, Cl12.55.

Since BU-2867T A was negative to ninhydrin and did not exhibit anytitratable group, its structure was logically that constructed bylinking VII and VIII by a peptide bond.

On heating with 6N HCl, both BU-2867T B and C afforded the same aminoacid complex as that obtained from BU-2867T A. The fatty acid moietiesof the two antibiotics were extracted from the hydrolyzate, treated withdiazomethane and isolated as methyl esters, IX (from BU-2867T B) and X(from BU-2867T C). They retained the characteristic UV maximum (λ_(max)^(MeOH) : 260 nm) observed for their parent antibiotics. The EI-MS of IXyielded a molecular ion at m/z 236, suggesting a C₁₄ -acid ester havingthree double bonds. The UV and ¹ H-NMR spectra distinctly showed 2(E),4(E)-dienoic acid structure and the third double bond to be isolated inIX. Ozonolysis of IX followed by reductive degradation of the ozonideyielded n-hexanal which was isolated and identified as2,4-dinitrophenyl-hydrazone (EI-MS M⁺ : m/z 280). These evidencesestablished that IX was methyl 2(E), 4(E), 8-tetradecatrienoate. Themolecular weight of ester X (EI-MS; M⁺ m/z 238) was found to be 28 units(C₂ H₄) higher than that of VI reflecting the molecular formuladifference observed between BU-2867T A and C. This, combined with the ¹H-NMR and UV data, revealed X to be methyl 2(E), 4(E)-tetradecadienoate.The results of the above experiments indicated the following structuresfor BU-2867T A, B and C:

    __________________________________________________________________________     ##STR7##                                                                 

    __________________________________________________________________________    BU-2867T A:      R = CH.sub.3(CH.sub.2).sub.6                                 BU-2867T B:      R = CH.sub.3(CH.sub.2).sub.4CHCH(CH.sub.2).sub.2             BU-2867T C:      R = CH.sub.3(CH.sub.2).sub.8                                 __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Physico-chemical Properties of BU-2867T A, B and C                                       BU-2867T A BU-2867T B BU-2867T C                                   __________________________________________________________________________    Nature     Colorless needles                                                                        White powder                                                                             Colorless needles                            M.p.       259-261° C.                                                                       232- 234° C.                                                                      273-275° C.                           [α].sub.D.sup.24° (c 0.5 MeOH)                                              -111°                                                                             -92°                                                                              -104°                                 Microanalysis:                                                                Calcd for  C.sub.27 H.sub.44 N.sub.4 O.sub.6.1/2H.sub.2 O                                           C.sub.29 H.sub.46 N.sub.4 O.sub.6.3/2H.sub.2                                             C.sub.29 H.sub.48 N.sub.4 O.sub.6                       C61.22, H8.56, N10.58                                                                    C60.71, H8.61, N9.77                                                                     C63.48, H8.82, N10.21                        Found      C60.90, H8.65, N10.47                                                                    C60.89, H8.31, N9.23                                                                     C63.48, H8.91, N10.16                        EI-MS m/z  520 (M.sup.+)                                                                            546 (M.sup.+)                                                                            548 (M.sup.+)                                UV λ .sub.max.sup.MeOH NM (ε)                                             261 (35,000)                                                                             261 (28,000)                                                                             261 (36,000)                                 TLC        0.45       0.41        0.34                                        Silanized plate Rf                                                            EtOH--H.sub.2 O (55:45)                                                       HPLC       6.43       7.93       11.33                                        Lichrosorb Rp-18 Rt                                                           EtOH--H.sub.2 O (65:35)                                                       __________________________________________________________________________

Enzymatic degradation of BU-2867T with Pseudomonas acylase gavedifferent products than those obtained by hydrolysis with ficin orpapain described above. Pseudomonas strain Pa-129 was fermented in 10 Lof medium containing 2% soluble starch, 0.2% glucose, 3% soybean meal,1% CaCO₃ and 0.3% MgSO₄. 7H₂ O at 37° C. for 3 days and the cells werecollected by centrifugation. After being washed with saline (1 L) twotimes, the cells were resuspended in 0.75 L of saline. The cellsuspension was mixed with pre-autoclaved suspension (1.5 L) of sodiumalginate (75 g) and CM-cellulose (75 g) and the mixture poured into 30 Lof 0.1M CaCl₂ solution under stirring. The gel entrapped in the cellswas stiffened by stirring with 25% glutaraldehyde solution and packed ina column (4.0×175 cm). A solution of BU-2867T A (1.5 g) in 20% aqueousmethanol (30 L) was passed through the column at a flow rate of 0.4-0.8L/hour. The pooled effluent was then passed through an Amberlite IRC-50(70% NH₄ ⁺ form, pH 6.7, 300 ml) and HP-20 column (300ml) successively.The IRC-50 column was washed with water and then developed with 1.5N NH₄OH. The ninhydrin positive fractions were pooled, concentrated andlyophilized to give pale yellow solid (800 mg) which was charged on acolumn of reverse phase silica (C₁₈, 250 ml). The column was developedwith water under medium-pressure and the ninhydrin-positive eluates werepooled and concentrated to yield white solid of L-threonylcyclic amine(XI, 612 mg). Yield 62%. M.p. 170° C., [α]_(D) ²⁷ -157° (c 0.5, H₂ O).EI-MS: m/z 342 (M⁺). UV: end adsorption. IR υ_(max) (KBr) cm⁻¹ : 3350,3280, 1650, 1620, 1530 etc. ¹ H-NMR (80 MH_(z), DMSO-d₆) δ 1.05 (3H, d),1.21 (3H, d), 1.4-2.2 (4H), 4.35 (2H, m), 4.47 (1H, m, OH), 4.62 (1H, d,OH), 6.16 (1H, d), 6.42 (1H, d-d), 7.36 (1H, t, NH), 7.97 (1H, d, NH),8.62 (1H, d, NH). ##STR8## The HP-20 column obtained above was developedwith water (1 L) and then a mixture of 0.1N NaOH and methanol (1:2).Fractions containing 2,4-dodecadienoic acid (V) were pooled,concentrated to 300 ml and acidified to pH 2.0. This solution wasextracted with ethyl acetate (300 ml) and n-butanol (100 ml).Evaporation of the ethyl acetate gave an oily residue which waschromatographed on a column of Sephadex LH-20 (800 ml). Upon elutionwith methanol and monitoring the eluates by UV absorption at 260 nm, theappropriate eluates were concentrated to yield colorless plates of pureV (259 mg). Yield 53%. M.p. 48°-49° C. UV λ_(max) ^(MeOH) nm (ε) : 258(24,000). IR υ_(max) (KBr) cm⁻¹ : 1680, 1630, 1605, 1410, 1300 etc. ¹H-NMR (80 MHz, CD₃ OD) δ 0.89 (3H, t), 2.0 (10H), 2.12 ( 2H, m), 5.75(1H, d), 6.16 (2H, m), 7.21 (1H, m). This compound was identical with2,4-dodecadienoic acid obtained by acid hydrolysis of BU-2867T A. Then-butanol extract was concentrated in vacuo and lyophilized to recoverthe starting material, BU-2867T A (160 mg, 11%).

It is apparent from the discussion above that compounds VIII and XI arevaluable intermediates useful in preparing the various antibioticcompounds of the present invention. Conventional acylation, for example,with a mixed anhydride of a carboxylic acid with an alkyl acid carbonate(HOCOOR) forms the requisite peptide bond, Advanced Organic Chemistry,Fieser & Fieser, pages 1039-1046, Reinhold Publishing Corporation, NewYork, 1965. Hydroxyl groups may be protected in any convenient manner,for example, as the tetrahydropyranyl or t-butyl ethers. Acylation ofthe amine group of VIII with the acids ##STR9## yields BU-2867T A, B andC, respectively. For example, BU-2867T A is prepared by the coupling ofVII and VIII. A mixture of VII (15 mg), N,N'-dicyclohexylcarbodiimide(10 mg) and 1-hydroxy-1,2,3-benzotriazole (8 mg) in 2 ml ofdimethylformamide was stirred for two hours at room temperature.Compound VIII (10 mg) was then added to the mixture and stirringcontinued overnight. The solution was concentrated to a residue whichwas chromatographed on reverse phase silica (C₁₈, 40 ml) with 80%methanol elution. The active eluates were evaporated and the residue waspurified by preparative HPLC (column: SSD-ODS-842, mobile phase: 90%aqueous methanol). Evaporation of the active peak fraction gave BU-2867TA (7.4 mg, yield 28%) which was identical in all respects with thenatural antibiotic. TLC (Silanized, EtOH-H₂ O=55:45) Rf: 0.45. HPLC(Lichrosorb RP-18, EtOH-H₂ O=65:35) Rt: 6.43 min.

BU-2867T A can also be prepared by coupling V with XI. Adimethylformamide solution (1 ml) of V (3.4 mg),N,N'-dicyclohexycarbodiimide (3.7 mg) and 1-hydroxy-1,2,3-benzotriazole(2.8 mg) was stirred for 2 hours at room temperature. To the solutionwas added XI (5 mg) and the mixture was kept stirring for additional 3hours and concentrated in vacuo. The residue was dissolved in methanoland purified by preparative HPLC using the same column and mobile phaseas described above. BU-2867T A 4 mg, yield 45%. Identity with naturalBU-2867T A was confirmed by a direct comparison.

Compound XI can be prepared from Compound VIII using conventionalprocedures. To a stirred mixture of N-t-butoxy-carbonyl-L-threonine (44mg), N,N'-dicyclohexylcarbodiimide (40 mg) and1-hydroxy-1,2,3-benzotriazole (30 mg) in dimethylformamide (4 ml) wasadded VIII (40 mg) at room temperature. The mixture was concentrated invacuo to a residue which was chromatographed on a column of reversephase silica (C₁₈, 40 ml) with methanol and water mixture (ratios: 1:9to 2:3). The appropriate fractions were pooled, concentrated in vacuoand lyophilized to yield N-BOC-L-threonyl-VIII (=BOC-XI). 51 mg Yield,69%. νc=o^(KBr) : 1690 and 1640 cm⁻¹. ¹ H-NMR (80 MHz) δ_(ppm) inDMSO-d₆ : 1.00 (3H, d), 1.22 (3H, d), 1.35 (9H, s), 6.11 (1H, d), 6.33(1H, d-d), etc. A mixture of BOC-XI (36 mg) and formic acid (1 ml) wasstirred for 1 hour at room temperature. This was concentrated, dilutedwith water (1 ml), adjusted pH 10.0 and applied to a column of reversephase silica (C₁₈, 20 ml). Upon developing with water and monitoring theeluate with ninhydrin test, the appropriate eluates were combined andfreeze-dried to give white solid of XI. 25 mg Yield 88%. ν_(c=o) ^(KBr): 1650 cm⁻¹. ¹ H-NMR δ_(ppm) in DMSO-d₆ : 1.04 (3H, d), 1.22 (3H, d),6:14 (1H, d), 6.42 (1H, d-d), 7.35 (1H, t, NH), 7.98 (1H, d, NH), 8.65(1H, d, NH) etc. EI-MS: m⁺ m/z 342.

Thus, it is possible to prepare the antibiotic compounds of theinvention by reaction of compounds VIII and XI with the appropriateacids, whatever their source. Further, using compounds VIII and XI,compounds according to the invention, particularly those produced inhigher yields, can be used to prepare other compounds of the invention.For example, BU-2867T A, which is obtained in higher yields byfermentation, can be used to prepare BU-2867T B and BU-2867T C, byenzymatic hydrolysis and coupling with the desired acid, as illustratedabove.

The yield of BU-2867T and the distribution of the A, B and C fractionsobtained by fermentation, as described above, can be modified by addingvarious fats and oils and certain surface active agents to the medium inwhich Polyangium brachysporum K481-B101 is being cultivated. Forexample, the addition of soybean oil, corn oil, olive oil, hydrogenatedvegetable oil, lard and tallow to a lesser extent, increased the totalyield of BU-2867T obtained. Fats and oils having a high content oflinoleic acid (C_(18:2)), such as soybean oil, corn oil and lard, led toan increase in the preparation of BU-2867T B formed. Oils, such as oliveoil, having a high content of oleic acid (C_(18:1)) led to an increasein the proportion of BU-2867T C formed. Hydrogenated vegetable oils richin stearic acid (C_(18:0)) only increased the amount of BU-2867T Aformed.

Antimicrobial Activity:

The minimum inhibitory concentrations (MICs) of BU-2867T A, B and C weredetermined for various microorganisms by a serial agar dilution method.Nutrient agar (Eiken) was used for bacteria and Sabouraud dextrose agar(Difco) for fungi. The inoculum size was adjusted to 10⁴ CFU/ml forbacteria and 10⁵ -10⁷ CFU/ml for fungi.

Table 5 shows in vitro antibacterial activities of BU-2867T A, B and C.Components A and C did not inhibit the growth of both gram-positive andgram-negative bacteria at 50 mcg/ml, while component B showed moderateactivity against some gram-positive bacteria.

The antifungal activities of BU-2867T components are shown in Table 6.Components C showed potent antifungal activity against clinicallyimportant pathogenic fungi such as Candida albicans, Cryptococcusneoformans, Aspergillus fumigatus, Trichophyton mentagrophytes and Mucorspinosus. Components A and B were somewhat less active than component C,but their anti-fungal spectra were similar to that of the latter.

                                      TABLE 5                                     __________________________________________________________________________    Antibacterial Spectra of BU-2867T Components                                                      MIC (mcg/ml)                                                                  BU-2867TA                                                                            BU-2867B                                                                            BU-2867C                                     __________________________________________________________________________    Gram-positive Organism                                                        Staphylococcus aureus                                                                       209P  >50    25    >50                                            "           Smith >50    3.1   >50                                            "           Bx-1633                                                                             >50    50    >50                                          Staphylococcus                                                                              D153  >50    6.3   >50                                          epidermidis                                                                   Staphylococcus                                                                              A22152                                                                              >50    50    >50                                          epidermidis                                                                   Streptococcus faecalis                                                                      A9612 >50    >50   >50                                          Micrococcus luteus                                                                          PCI-1001                                                                            >50    50    >50                                          Bacillus subtilis                                                                           PCI-219                                                                             >50    50    >50                                          Gram-negative Organism                                                        Escherichia coli                                                                            NIHJ  >50    >50   >50                                          Klebsiella pneumoniae                                                                       D-11  >50    >50   >50                                          Proteus mirabilis                                                                           A9554 >50    >50   >50                                          Proteus vulgaris                                                                            A9436 >50    >50   >50                                          Morganella morganii                                                                         A9553 >50    >50   >50                                          Serratia marcescens                                                                         A20222                                                                              >50    >50   >50                                          Enterobacter cloacae                                                                        A9659 >50    >50   >50                                          Pseudomonas aeruginosa                                                                      A9930 >50    >50   >50                                          __________________________________________________________________________     Medium: Nutrient agar                                                    

                                      TABLE 6                                     __________________________________________________________________________    Antifungal Spectra of BU-2867 T Components                                                         MIC (mcg/ml)                                             Test organism        BU-2867A                                                                            BU-2867B                                                                            BU-2867C                                     __________________________________________________________________________    Candida albicans                                                                            IAM 4888                                                                             3.1   3.1   1.6                                            "           A 9540 1.6   1.6   0.8                                          Cryptococcus  D 49   25    25    3.1                                          neoformans                                                                    Cryptococcus  IAM 4514                                                                             25    25    3.1                                          neoformans                                                                    Aspergillus fumigatus                                                                       IAM 2530                                                                             1.6   3.1   0.8                                            "           IAM 2034                                                                             1.6   3.1   0.8                                          Aspergillus flavus                                                                          FA 21436                                                                             25    25    50                                           Fusarium moniliforme                                                                        A 2284 >50   >50   >50                                          Piricularia oryzae                                                                          D 91   >50   >50   >50                                          Trichophyton  D 155  25    12.5  1.6                                          mentagrophytes                                                                Trichophyton  #4329  25    12.5  6.3                                          mentagrophytes                                                                Blastomyces dermaditis                                                                      IFO 8144                                                                             50    50    >50                                          Sporothrix schenckii                                                                        IFO 8158                                                                             >50   >50   >50                                          Petriellidium boydii                                                                        IFO 8073                                                                             >50   >50   50                                           Mucor spinosus                                                                              IFO 5317                                                                             1.6   0.8   0.2                                          __________________________________________________________________________     Medium: Sabouraud dextrose agar                                          

Antitumor activity:

The antitumor activity of BU-2867T A, B and C was determined in femaleCDF₁ and male BDF₁ mice. Lymphocytic leukemia P388 (CDF₁ and BDF₁ mice)and lymphoid leukemia L1210 (BDF₁ mice) were inoculated byintraperitoneal injection of 0.8 ml diluted ascitic fluid containing 10⁶and 10⁵ cells per mouse, respectively. Melanotic melanoma B16 (BDF₁mice) were implanted 0.5 ml of a 10% tumor brei intraperitoneally. Testmaterials were dissolved in 0.9% saline containing 10% dimethylsulfoxide and graded doses of them were administered to miceintraperitoneally 24 hours after tumor implantation. Either olivomycin(NSC 38270) or mitomycin C was comparatively tested as a referencecompound in the experiments.

BU-2867T A, B and C showed antitumor activity against P388 leukemia bythe treatment schedule 1 (once daily on days 1, 4 and 7). Table 7 showsthe results obtained as the increase in median survival time (MST) oftest (T) and control (C) animals for various dosage regimens expressedas a percentage ratio. Values for percentage ratios of 125 and aboveindicate significant antitumor effect.

                  TABLE 7                                                         ______________________________________                                        Antitumor Activity of BU-2867T Components                                     Against P388 Leukemia                                                                    T/C % of MST                                                                  Dose in mg/kg/day, ip                                                         3    1          0.3    0.1                                         ______________________________________                                        BU-2867T A          140        130  120                                       BU-2867T B   165    140        120                                            BU-2867T C   155               130                                            Olivomycin A 140    135        100                                            ______________________________________                                    

BU-2867T A and C were highly active against P388 leukemia by thetreatment schedule 2 (once daily on days 1 through 9); BU-2867T A wasless active against L1210 leukemia and B16 melanoma. Table 8 shows theresults obtained for various schedule dosage regimens again expressed asa percentage ratio increase in median survival time, with values of 125and above indicating significant antitumor effect.

                  TABLE 8                                                         ______________________________________                                        Antitumor Activity of BU-2867T A and C                                                 T/C% of MST                                                                   Dose in mg/kg/day, ip                                                         2     1      0.5    0.25 0.13 0.063                                                                              0.031                             ______________________________________                                        P388 leukemia                                                                 BU-2867T A 67      228    186  156  147  136  109                             BU-2867T C 234     189    175  159  153  123  100                             Mitomycin C        290    240  170  150  130  115                             L1210 leukemia                                                                BU-2867T A                129  118  118  106                                  Mitomycin C        140    141  129  129  106                                  B16 melanoma                                                                  BU-2867T A Tox.    125    116  113  100                                       Mitomycin C                                                                              63      181    163  141  131                                       ______________________________________                                    

The acute toxicity of BU-2867T A and C was determined in male ddY miceby single intraperitoneal administration. The LD₅₀ values were 8.1 mg/kgand 25 mg/kg, respectively.

The pharmacologically effective compounds of this invention can beprocessed by conventional methods of galenic pharmacy intopharmaceutical preparations for oral or parenteral administration, e.g.,to mammals including humans. Conventional excipients arepharmaceutically acceptable organic or inorganic carrier substancesuitable for parenteral, enteral or topical application which do notdeleteriously react with the active compounds. Suitable pharmaceuticallyacceptable carriers include, but are not limited to , water, saltsolutions, alcohols, gum arabic, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,petrolatum, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy-methylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and,if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, flavoring and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

For parenteral application, particularly suitable are injectable sterilesolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants, including suppositories. Ampoules are convenientunit dosages.

For enteral application, particularly suitable are tablets, dragees,suppositories or capsules having talc and/or a carbohydrate carrier orbinder or the like, the carrier preferably being lactose and/or cornstarch and/or potato starch. A syrup, elixir or the like can be usedwherein a sweetened vehicle is employed. Sustained release compositionscan be formulated including those wherein the active compound isprotected with differentially degradable coatings, e.g., bymicroencapsulation, multiple coatings, etc.

Generally, the compounds of this invention are dispensed in unit dosageform in a pharmaceutically acceptable carrier comprising the requisitedosage. The dosage of the compounds according to this inventiongenerally is 5-250 mg/day when administered to patients, e.g., humansweighing 75 kg. Suitable dosages and regimens for a given host can bedetermined using conventional considerations, e.g., by customarycomparison of the differential activities of the subject compound and ofa known antibiotic or antitumor agent, e.g., by means of an appropriate,conventional pharmacological protocol.

From the foregoing description, one skilled in the art can readilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A method for the preparation of a compound of theformula ##STR10## wherein R is CH₃ --(CH₂)₆, CH₃ --(CH₂)₄--CH═CH--(CH₂)₂ or CH₃ --(CH₂)₈, which comprises culturing Polyangiumbrachysporum sp. nov. strain K481-B101 (ATCC 53080) in a nutrient mediumcontaining an assimilable source of carbon and nitrogen under aerobicconditions, separating the mycelia produced and recovering the compoundfrom the nutrient medium.
 2. A method according to claim 1 wherein a fator oil is added to the nutrient medium.
 3. A method according to claim 1wherein the separated mycelia are extracted with an organic solvent andthe extract combined with the nutrient medium prior to recovery of thecompound.
 4. A method according to claim 1 wherein the compound isrecovered from the nutrient medium by extraction with an organicsolvent.
 5. A method according to claim 1 wherein production of thecompound is monitored using Candida albicans as the test organism.
 6. Abiologically pure culture of the microorganism Polyangium brachysporumsp. nov. strain K481-B101 (ATCC 53080).